Detailed localization of aquaporin-4 messenger RNA in the CNS: preferential expression in periventricular organs

We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.     

Expression of neurotrophins in hippocampal interneurons immunoreactive for the neuropeptides somatostatin, neuropeptide-Y, vasoactive intestinal polypeptide and cholecystokinin.

Using a double detection method, which combines in situ hybridization for the detection of neurotrophin messenger RNA with immunocytochemistry against the neuropeptides somatostatin, neuropeptide Y, vasoactive intestinal polypeptide and cholecystokinin, we have analysed the expression of the neurotrophins, nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, in distinct populations of neuropeptide-immunoreactive hippocampal interneurons. Nerve growth factor messenger RNA expression was found in subsets of the four subpopulations of neuropeptide-immunoreactive interneurons. The highest degree of co-localization was observed in the neuropeptide-Y-positive cells (up to 70%) and in somatostatin-immunoreactive cells (48%). Only small subsets of cholecystokinin- and vasoactive intestinal polypeptide-positive neurons (21% and 10%, respectively) displayed nerve growth factor hybridization signals. In contrast, expression of neurotrophin-3 messenger RNA was exclusively observed in 26% of neuropeptide-Y-immunoreactive cells. Brain-derived neurotrophic factor hybridization signals were never detected in the neuropeptide-positive hippocampal interneurons. Morphological analysis of neuropeptide-immunoreactive interneurons that express or lack nerve growth factor messenger RNA revealed that most perisomatic inhibitory neurons, such as large vasoactive intestinal polypeptide/ cholecystokinin-immunoreactive cells, showed positive nerve growth factor hybridization signals. In addition, some somatostatin/neuropeptide-Y-immunoreactive interneurons, which are responsible for dendritic inhibition of principal hippocampal neurons, expressed nerve growth factor messenger RNA. In contrast, interneurons specialized to innervate other GABAergic cells, such as small vasoactive intestinal polypeptide-positive cells, lacked nerve growth factor expression. All these data indicate that expression of neurotrophins is differentially regulated in functionally distinct classes of hippocampal interneurons immunoreactive for neuropeptides. We also analysed whether neuropeptide-immunoreactive interneurons expressing neurotrophins were targets of the GABAergic septohippocampal pathway. We used a triple detection method, combining anterograde tracing of this connection, with in situ hybridization for the detection of neurotrophin mRNA, and immunocytochemistry against neuropeptides. Our data showed that the four populations of hippocampal interneurons studied (somatostatin, neuropeptide-Y, vasoactive intestinal polypeptide and cholescystokinin) received GABAergic afferents from the septum. However, no preference for neuropeptide-immunoreactive cells expressing neurotrophins was observed, compared to neuropeptide-positive neurons lacking neurotrophin expression.     

Cellular and regional expression of glutamate dehydrogenase in the rat nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry.

In the central nervous system glutamate dehydrogenase appears to be strongly involved in the metabolism of transmitter glutamate and plays a role in the pathogenesis of neurodegenerative disorders. In order to identify unequivocally the neural cell types expressing this enzyme, non-radioactive in situ hybridization, using a complementary RNA probe and oligonucleotide probes, was applied to sections of the rat central nervous system and, for comparison with peripheral neural cells, to cervical spinal ganglia. The results were complemented by immunocytochemical studies using a polyclonal antibody against purified glutamate dehydrodenase. Glutamate dehydrogenase messenger RNA was detectable at varying amounts in neurons and glial cells (i.e. astrocytes, oligodendrocytes, Bergmann glia, ependymal cells, epithelial cells of the plexus choroideus) throughout the central nervous system and in neurons and satellite cells of spinal ganglia. In some neuronal populations (e.g., pyramidal cells of the hippocampus, motoneurons of the spinal cord and spinal ganglia neurons) messenger RNA-labelling was higher than in other central nervous system neurons. This is remarkable because the immunostaining of neurons in the central nervous system regions studied was at best weak, whereas a predominantly high level of immunoreactivity was detected in astrocytes (and Bergmann glia). Thus, in neurons of the central nervous system, the detected levels of glutamate dehydrogenase messenger RNA and protein seem to be at variance whereas in peripheral neurons of spinal ganglia both in situ hybridization labelling and immunostaining are intense.     

Evidence for the existence of non-GABAergic, cholinergic interneurons in the rodent hippocampus

Previous studies have revealed a small number of hippocampal interneurons immunoreactive for choline acetyltransferase, the acetylcholine-synthesizing enzyme. It remained an open question, however, whether these neurons represented a subgroup of inhibitory GABAergic neurons co-localizing acetylcholine. In this study, we have combined immunocytochemistry for choline acetyltransferase and in situ hybridization for glutamate decarboxylase messenger RNA, the GABA-synthesizing enzyme. None of the choline acetyltransferase-immunoreactive neurons in the various layers of the hippocampus proper and fascia dentata were found to co-localize glutamate decarboxylase messenger RNA. The lack of an in situ hybridization signal in these neurons is unlikely to result from the combination of the two labeling techniques. When combining in situ hybridization for glutamate decarboxylase messenger RNA with immunostaining for parvalbumin, a calcium-binding protein expressed by many GABAergic hippocampal interneurons, numerous double-labeled cells were observed. These data provide neurochemical evidence for the existence of non-GABAergic, supposedly cholinergic non-principal cells in the hippocampus.     

The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system.

Cation-chloride cotransporters have been considered to play pivotal roles in controlling intracellular and extracellular ionic environments of neurons and hence controlling neuronal function. We investigated the total distributions of K-Cl cotransporter 1 (KCC1), KCC2 (KCC2), and Na-K-2Cl cotransporter 1 (NKCC1) messenger RNAs in the adult rat nervous system using in situ hybridization histochemistry. KCC2 messenger RNA was abundantly expressed in most neurons throughout the nervous system. However, we could not detect KCC2 messenger RNA expression in the dorsal root ganglion and mesencephalic trigeminal nucleus, where primary sensory neurons show depolarizing responses to GABA, suggesting that the absence of KCC2 is necessary for this phenomenon. Furthermore, KCC2 messenger RNA was also not detected in the dorsolateral part of the paraventricular nucleus, dorsomedial part of the suprachiasmatic nucleus, and ventromedial part of the supraoptic nucleus where vasopressin neurons exist, and in the reticular thalamic nucleus. As vasopressin neurons in the suprachiasmatic nucleus and neurons in the reticular thalamic nucleus produce their intrinsic rhythmicity, the lack of KCC2 messenger RNA expression in these regions might be involved in the genesis of rhythmicity through the control of intracellular chloride concentration. The expression levels of KCC1 and NKCC1 messenger RNAs were relatively low, however, positive neurons were observed in several regions, including the olfactory bulb, hippocampus, and in the granular layer of the cerebellum. In addition, positive signals were seen in the non-neuronal cells, such as choroid plexus epithelial cells, glial cells, and ependymal cells, suggesting that KCC1 and NKCC1 messenger RNAs were widely expressed in both neuronal and non-neuronal cells in the nervous system.These results clearly indicate a wide area- and cell-specific variation of cation chloride cotransporters, emphasizing the central role of anionic homeostasis in neuronal function and communication.     

Localization of central cannabinoid CB1 receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: a double-label in situ hybridization study

In situ hybridization histochemistry was used to show the distribution of messenger RNA for central cannabinoid CB 1 receptors in dorsal root ganglia of the rat. CB1 messenger RNA was highly expressed in neuronal subpopulations of rat dorsal root ganglia. The phenotypes of neurons that express messenger RNA for CB1 were subsequently examined by combining a 35S-labeled ribonucleotide probe for CB1 messenger RNA with digoxigenin-labeled riboprobes for preprotachykinin A (substance P precursor), alpha-calcitonin gene-related peptide and preprosomatostatin (somatostatin precursor) messenger RNAs. Qualitative examination revealed expression of CBI messenger RNA predominantly in medium-and large-sized cells distributed throughout the dorsal root ganglia. The majority of neurons expressing substance P messenger RNA were CB1 messenger RNA negative and smaller in size than the CB1 messenger RNA-positive cells. Only 13% of substance P messenger RNA-positive cells expressed CB1 messenger RNA. A similar degree of co-localization was observed with alpha-calcitonin gene-related peptide: 10% of cells expressing messenger RNA for this neuropeptide were CB1 messenger RNA positive. Co-localization of CB1 and somatostatin messenger RNAs was observed in less than 0.5% of somatostatin messenger RNA-positive cells. The data suggest that subpopulations of neurons in rat dorsal root ganglia are capable of synthesizing cannabinoid receptors and inserting them on terminals in the superficial dorsal horn. These findings provide anatomical evidence for cannabinoid modulation of primary afferent transmission. Although an anatomical basis for cannabinoid-mediated suppression of release of neurogenic peptides from nociceptive primary afferents is provided, our results demonstrate that the majority of CB messenger RNA-positive neurons in the dorsal root ganglia contain transmitters and/or neuromodulators other than the neuropeptides examined herein.     

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.     

The cellular localization of preprotachykinin A messenger RNA in the bovine nervous system

The cellular distribution of preprotachykinin A messenger RNA in the bovine nervous system was investigated by in situ hybridization and its tissue distribution by Northern and dot blotting. The latter results were compared with the levels of substance P-like immunoreactivity as determined by radio-immunoassay. The highest levels of preprotachykinin A messenger RNA were found in striatum and trigeminal ganglion, medium levels in retina and lower levels in hypothalamus, spinal cord, pituitary gland and adrenal medulla. The cellular localization of preprotachykinin A messenger RNA was obtained in striatum and trigeminal ganglion using either single-stranded DNA or complementary RNA probes labelled with 32P, 35S or 3H. Specific labelling of small trigeminal ganglion neurones and of medium-sized striatal nerve cells was observed with probes in the anti-messenger RNA sense orientation. Only background labelling was obtained with probes in the messenger RNA sense orientation. The technique was further validated by the demonstration that the same cells in the trigeminal ganglion were labelled by both in situ hybridization and immunohistochemistry. The present findings allow an unambiguous identification of the cellular sites of synthesis of preprotachykinin A messenger RNA; in situ hybridization should also prove a useful technique for investigating the regulation of neuropeptide biosynthesis at the cellular level.     

Intraregional variation in expression of serotonin transporter messenger RNA by 5-hydroxytryptamine neurons

The distribution of the messenger RNA encoding the 5-hydroxytryptamine transporter was investigated in rat brain. 5-Hydroxytryptamine transporter messenger RNA was found exclusively in the B1-B9 cell groups containing the cell bodies of 5-hydroxytryptamine neurons. Combined in situ hybridization and 5-hydroxytryptamine immunocytochemistry demonstrated 5-hydroxytryptamine transporter gene expression in the majority of and exclusively in 5-hydroxytryptamine neurons. Cells differed in their levels of expression of 5-hydroxytryptamine transporter messenger RNA and 5-hydroxytryptamine immunofluorescence, but with a tight correlation between the two parameters. Image analysis of cells from B7, the dorsal raphe nucleus, and B8, the median raphe nucleus, revealed significant differences between groups in the mean cellular level of 5-hydroxytryptamine transporter gene expression. Cells in the ventromedial subdivision of B7 displayed higher levels of expression than cells in B8 or cells in the lateral wings of B7. There was also heterogeneity in the distribution of the cellular levels of expression for two other genes expressed by 5-hydroxytryptamine neurons: l-aromatic amino acid decarboxylase messenger RNA and tryptophan hydroxylase messenger RNA. However, the relative levels of expression of these two genes within the four regions studied differed from that of 5-hydroxytryptamine transporter messenger RNA. These results indicate intraregional differences between 5-hydroxytryptamine neurons with respect to 5-hydroxytryptamine transporter messenger RNA levels. Such differences may account for the differential sensitivity of 5-hydroxytryptamine neurons to cytotoxins.     

Calretinin distribution in the thalamus of the rat: immunohistochemical and in situ hybridization histochemical analyses.

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei. A subpopulation of calretinin cell bodies was also identified in the reticular nucleus. The mediorostral lateral posterior nucleus, subparafascicular, lateral geniculate and habenular nuclei also contained calretinin messenger RNA probe label. In contrast, no positive cells were found in the anterior, ventral or posterior thalamic nuclei. The distribution of calretinin cells did not correspond directly with that of other histochemical markers. Thus, the in situ hybridization histochemical and immunohistochemical results revealed calretinin as a unique identifying marker for distinct sets of thalamic neurons.     

Neuronal nitric oxide synthase (bNOS) messenger RNA and protein expression in developing rat brainstem nuclei

Neuronal nitric oxide synthase (bNOS) messenger RNA expression and immunoreactivity were mapped in series of cryosections through the developing rat brainstem nuclei. Between embryonic day E16 and postnatal day P16, brainstem nuclei expressed both bNOS messenger RNA (mRNA) in situ hybridization signals and protein immunoreactivity. However, NOS mRNA signals were absent from the Edinger Westphal, facial or motory trigeminal nucleus. Strong patterns of mRNA signals and immunoreactivity occurred in neurons located in the substantia nigra pars compacta and the laterodorsal tegmental nuclei. Between E24 and P16, altered patterns of bNOS mRNA positive and immunoreactive neurons, e.g. superior and inferior colliculi, raphe nuclei, solitary tract or pontine nucleus were documented. Altered NOS expression patterns thus may reflect developmental processes within distinct neuronal populations such as cell phenotype discrimination or synaptogenesis within efferent or afferent brainstem pathways. The NOS/NO system therefore appears to be a modulator for intra-/intercellular adjustment processes in normal development.     

Unilateral dopamine depletion increases expression of the 2A subunit of the N-methyl-D-aspartate receptor in enkephalin-positive and enkephalin-negative neurons

Striatal efferent neurons receive dopamine- and glutamate-utilizing afferents. Previous studies have shown that dopamine depletion increases gene expression in striatopallidal neurons and decreases it in striatonigral neurons. Previous work has also reported increased expression of the 2A subunit of the N-methyl-D-aspartate receptor in the dopamine-depleted striatum. The purpose of this study therefore was to determine whether dopamine depletion differentially alters the expression of the 2A subunit of the N-methyl-D-aspartate receptor in rat striatal neurons. 6-Hydroxydopamine (8microg/2microl) was infused unilaterally into the medial forebrain bundle. Rats were killed three weeks later. Double-label in situ hybridization was performed using an 35S-labeled ribonucleotide probe directed against the messenger RNA of the 2A subunit and a digoxigenin-labeled ribonucleotide probe directed towards preproenkephalin messenger RNA to mark striatopallidal neurons. Analysis of single-labeled film autoradiograms revealed a significant increase in the expression of 2A subunit messenger RNA in the ipsilateral, but not the contralateral, striatum of dopamine-depleted animals, consistent with other studies in the literature. Cellular analysis of 2A subunit expression indicated that as a consequence of dopamine depletion there is a significant increase in the expression of this subunit in both enkephalin-positive and enkephalin-negative neurons. From this study we conclude that dopamine depletion increases messenger RNA expression of the 2A subunit of the N-methyl-D-aspartate receptor in striatopallidal and presumed striatonigral (enkephalin-negative) neurons. Such alterations may affect the pharmacology and function of the resultant receptor, and thus alter glutamate transmission in both populations of medium spiny neurons after dopamine depletion.     

Bcl-2, Bax and Bcl-x expression following kainic acid administration at convulsant doses in the rat.

Neuronal death was produced in the CA1 and CA3 areas of the hippocampus, amygdala, and piriform and entorhinal cortices after intraperitioneal administration of kainic acid at convulsant doses to adult rats. To assess the involvement of members of the Bcl-2 family in cell death or survival, immunohistochemistry, western and northern blotting to Bcl-2, Bcl-x and Bax, and in situ hybridization to Bax were examined at different time-points after kainic acid treatment. Members of the Bcl-2 family were expressed in the cytoplasm of pyramidal neurons in the hippocampus, and in a subset of neurons of the piriform and the entorhinal cortices, amygdala and neocortex in the normal adult brain. Dying neurons in the pyramidal cell layer of CA1 and CA3 areas, entorhinal and piriform cortices, and amygdala also expressed Bcl-2, Bax and Bcl-x following excitotoxicity, although many dying cells did not. In addition, a number of cells in the affected areas showed Bax immunoreactivity in their nuclei at 24-48 h following kainic acid administration, thus indicating Bax nuclear translocation in a subset of dying cells. Western blots disclosed no modifications in the intensity of the bands corresponding to Bcl-2, Bcl-x and Bax, between control and kainic acid-treated rats. No modifications in the intensity of the bcl-2 messenger RNA band on northern blots was observed in kainic acid-treated rats. However, a progressive increase in the intensity of the bax messenger RNA band was found in kainic acid-treated rats at 6 h, 12 h and 24 h following kainic acid administration. Interestingly, a slight increase in Bax immunoreactivity was observed in the cytoplasm of neurons of the dentate gyrus at 24-48 h, a feature which matches the increase of bax messenger RNA in the same area, as shown by in situ hybridization at 12-24 h following kainic acid injection. The present results suggest that cell death or survival does not correlate with modifications of Bcl-2, Bax and Bcl-x protein, and messenger RNA expression, but rather that kainic acid excitotoxicity is associated with Bax translocation to the nucleus in a subset of dying cells.     

Galectin-1 and galectin-3 in chronic pancreatitis

Galectin-1 and galectin-3 have important functions in cell-cell interactions, cell adhesion to extracellular matrix, the organization of extracellular matrix, and tissue remodeling. To assess their potential role in chronic pancreatitis (CP), we examined their expression by Northern blot analysis, in situ hybridization, immunohistochemistry, and Western blot analysis in normal and CP pancreatic tissues. Northern blot analysis revealed a 4.5-fold increase of galectin-1 mRNA (p < 0.01) and a 3.8-fold increase of galectin-3 mRNA (p < 0.01) in CP samples compared with normal controls. In situ hybridization analysis of normal pancreas indicated low abundance of galectin-1 mRNA in fibroblasts, whereas galectin-3 mRNA was moderately present in ductal cells. CP samples exhibited moderate to intense galectin-1 mRNA signals in fibroblasts, whereas galectin-3 mRNA signals were intense in the cells of ductular complexes and weak in the degenerating acinar cells. In addition, intense galectin-1 and galectin-3 mRNA signals were present in nerves of normal and CP samples. Immunohistochemistry showed a distribution pattern of galectin-1 and galectin-3 similar to that described for in situ hybridization. Relative quantification of galectin-1 and galectin-3 protein by immunoblotting revealed an increase of 3.2-fold and 3.0-fold, respectively, in CP compared with normal controls. There was a significant correlation between galectin-1 and fibrosis and between galectin-3 and fibrosis and the density of ductular complexes. Up-regulation of galectin-1 in fibroblasts and galectin-3 in ductular complexes suggests a role of these lectins in tissue remodeling in CP. Galectin-1 might participate in ECM changes, whereas galectin-3 seems to be involved in both ECM changes and ductular complex formation.     

Repeated electroconvulsive shock increases tachykinin and cholecystokinin mRNA expression in ventral periaqueductal gray.

The effect of repeated electroconvulsive shock (five shocks during 10 days) on preprocholecystokinin and preprotachykinin-A messenger RNA expression was studied in the mesencephalic periaqueductal gray and adjacent areas of rat using in situ hybridization histochemistry with specific oligonucleotide probes. An increased number of preprocholecystokinin and preprotachykinin-A messenger RNA hybridization positive neurons (+30% and +47%, respectively) in the Edinger-Westphal nucleus was observed following repeated electroconvulsive shock. In addition, both preprocholecystokinin and preprotachykinin-A messenger RNA expression, measured as grain density over single neurons, was significantly increased (+37% and +45%, respectively). The results indicate that cholecystokinin- and substance P-containing neurons in the Edinger-Westphal nucleus are activated by repeated electroconvulsive shock, which may be related to the antidepressant and analgesic effects of electroconvulsive shock treatment.